Card member for receiving a tissue specimen to be processed for histological examination

ABSTRACT

The present invention is directed to a card member for receiving a tissue specimen to be processed for histological examination including an absorbent portion for maintaining the tissue specimen in engageable contact with the card member, and a generally non-adhesive portion for allowing separation of the tissue specimen from the card member, where the card member provides the specimen with a known, consistent orientation throughout processing.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of prior U.S. patent application Ser.No. 11/001,305, filed Dec. 1, 2004, which is hereby incorporated hereinby reference in its entirety, which is a continuation-in-part of priorU.S. patent application Ser. No. 10/723,692, filed Nov. 26, 2003, whichis also hereby incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to an apparatus and method for preparing atissue specimen for histological examination and, more particularly, toan apparatus and method for processing the tissue specimen from the timeof collection of the specimen to the preparation of the microscopeslide.

BACKGROUND OF THE INVENTION

It is a standard procedure to prepare tissue samples for microscopicexamination by embedding the tissue in paraffin and slicing theparaffin-embedded tissue very thinly with a microtome. Preparatory toembedding, the tissue is treated in various solutions appropriate to itsexamination and long-term stability. Typically, prior to paraffinembedding, the tissue sample is fixed, dehydrated, and cleared and theninfiltrated with molten paraffin.

Typically, the tissue sample collected for examination is a unitary,connected portion of tissue, however, small parts of the tissue samplemay be dislocated during tissue processing. Alternatively, a biopsy maybe performed on minute fragments less than 1.0 mm in diameter, such asbronchial washings, cytology preparations and aspiration biopsies whichmay be gathered by skinny needles or imaging technology-guided tissuebiopsy devises. Generally, this technique is called “tissue processing,”and it includes the following: (1) collection of the specimen; (2)fixation of the specimen to preserve tissue components; (3) sampling ofa representative portion or aliquot; and (4) cutting of thenon-processed tissue in a section plane to be presented to the microtomeblade (for microscopic examination and information collection) andplacement of this plane face down in a tissue processing cassette forcontainment during the “tissue processing phase.”

Information obtained in the microscopic study of biological tissue istaken from microtome cut sections that, on average, are less than 10μthick. Thus, in view of the small size of the tissue sample and theprecision with which the microtome cut sections are cut, it is ofparamount importance to select a cutting plane and to maintain thisplane as closely as possible, if not exactly, during processing,embedding and sectioning. Further, in the instance of minute specimens(approximately 1 mm or less), it may also be desirable to preserve theorientation of the tissue sample in relation to the adjacent structuresfrom which it was collected. Such information may provide guidance forsurgical or other treatments.

Tissue placed in a processing cassette may be fresh or fixed. The tissuesample is then passed through fixatives to remove water from the sample.Following dehydration, the tissue sample is then processed with asolvent that will dissolve fatty materials and “clear” the tissuesample. After being “cleared”, the tissue sample is placed in moltenparaffin and it is infiltrated with the wax. Molten wax replaces thesolvent which will evaporate or be diluted to trace levels, causing allthe tissue to be infiltrated with a common wax binder.

Next, the cassette is opened and the infiltrated tissue is placed into ametal or similar base mold filled with melted wax. A considerable effortmust be made at this moment to cause the “face down” selected cuttingplane of the tissue to be positioned in the exact same planarrelationship that was selected by the doctor or technician who placedthe sample and selected the portion of the tissue that would be cut inmicrotomy. It is critical in many examinations, such as cancerdiagnosis, to maintain the parallel relationships of: (a) selectedcutting surface; (b) microtomy cut surface; (c) glass slide stained andcovered cut section; and (d) microscopic section orientation forexamination.

The technician thus must attempt to present the specimen to themicrotome cutting blade in the exact position previously selected. Thetechnician prepares the specimen for microscopic examination bysubsequently mounting, staining and cover-slipping the microtome cutsection on a microscope slide. Although it is desirable to maintain thepositioning of the specimen, it is very difficult to control theposition of the specimen particularly with minute tissue samples. Asmall (0.25-1.0 mm) specimen may likely shift its position in thecassette during processing or when it is removed from the processingcassette to the embedding mold prior to wax casting, so that thepre-selected position of the specimen may be lost.

Apparatuses are known that may be used for processing of tissue samples,such as described in U.S. Pat. Nos. 4,557,903 and 4,569,647, both toMcCormick. In both the '903 and '647 patents, the tissue sample isdeposited somewhat randomly in the cassette. It is a limitation that,although the tissue sample is disposed in an enclosed area, it may movefreely within that area as the tissue sample is contacted with solventsduring processing. Additionally, the apparatus of the '903 patentrequires removing the tissue sample from the cassette and placing it ina mold for casting in paraffin. A shortcoming of the '903 and '647patents is their inability to maintain the tissue sample in a desiredorientation throughout processing.

There remains a need for an apparatus and processing method that permitsthe tissue plane selected by the surgeon or pathologist to be processedin situ without disturbing the orientation of the tissue specimen. Moreparticularly, there is a need for an assembly and method that maintainsthe desired position of the tissue sample during the fixing, processing,and wax embedding steps, as well as during subsequent sectioning in amicrotome and mounting of the desired section on a glass slide forstaining, cover-slipping and microscopic examination. Such a systemshould be adaptable for both large specimen sections and minutefragments of less than 1 mm in size.

SUMMARY OF THE INVENTION

The present invention is directed to a system and method for use in thepreparation in situ of a tissue specimen for histological examination.The system and method allows a selected cutting plane of the specimen tobe maintained throughout processing, from the time the plane is selectedthrough sectioning of the specimen in the microtome to improve theaccuracy and effectiveness of histological examination. The systemincludes a card or positioning member for mounting the tissue specimenin the desired position that is maintained throughout processing, acassette or carrier member for receiving the card with the mountedtissue specimen and for subsequently mounting the processed tissuespecimen in a microtome, and a base mold having a lower recessed chamberfor receiving the card with mounted tissue specimen.

The mounting card may include an essentially non-adhesive surface, suchas metal or polyester, and a porous, absorbent underlayment material.The upper surface of the mounting card may include a thin film, such aspolypropylene, polytetrafluoroethylene, polyethylene, polyester andnylon films. Alternatively, the upper surface may include a thin (about0.004 to 0.006 inch thick) piece of metal, such as stainless steel, orplastic. The upper surface may be perforated to have a void spacesufficient to allow the denatured tissue juice to pass therethrough soas to form a protein bridge into the absorbent underlayment material.Or, in the instance of previously fixed tissue, the penetration andencapsulation by a molten agar or gel that when cool will process andembed for microtome sectioning.

The base mold may include an upper interior area for receiving thecassette and a lower chamber for receiving the mounting card withspecimen. The front and back walls may have lines of weakness to allowthe walls to be separated from the base mold to facilitate access to theinterior of the base mold when it is desired to remove the castingblock. The base mold also may include an opening in the lower chamber sothat a process line may be connected to the base mold. The base mold mayalso include a removable bottom wall.

In accordance with the method of the invention a tissue specimen iscollected and a desired cutting plane identified. The specimen isdisposed on the surface of a semi-rigid or rigid mounting card with thedesired cutting plane positioned downwardly on the surface of the card.A carbohydrate or protein-based glue or the like may be utilized inmounting the specimen on the card. As an example, such glue may be apolysaccharide, including 5% agar or polyamide gel. The card withapplied specimen may be placed in a cassette for transporting thespecimen. The specimen so mounted on a card within a cassette may beplaced in an appropriate container of tissue fixative for transport tothe processing laboratory. Alternatively, the specimen on the mountingcase may be covered with a fixative gel, such as 10% formalin in a resingel base. The specimen next may be processed with suitable fluidsnecessary for preparing the specimen for histological examination. Thespecimen on the card may be processed in the cassette or may be removedfrom the cassette and placed in a base mold for processing.

Subsequent to processing, the specimen card is placed in the base mold,if processing did not occur in the base mold, for embedding. The cardwith the specimen may be placed in a lower chamber of a base mold, whilethe cassette may be placed in an upper chamber of the base mold abovethe card. Embedding material, such as paraffin is introduced into thebase mold. Before the introduction of the embedding material, a vacuummay be drawn to flatten the card against the base mold to provide asmooth planar surface. Or, as an alternative method, the lower chamberof the mold may be open and a closure containing the specimen cardapplied to the opening thus positioning the card, specimen face up, inthe bottom chamber for wax casting.

The embedded specimen and cassette may be recovered from the base moldin the form of an investment casting tissue block which includes thecassette as the base of the block and an outwardly extending,specimen-containing examination portion of the block. The block may bemounted in a parallel relationship to the microtome by engaging thecassette with the microtome chuck, and the block may be sectioned by themicrotome blade as a planar section.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an exploded perspective view of an assembly for in situprocessing of a tissue sample embodying features of the presentinvention illustrating an exemplary carrier, positioning member, basemember, and tissue sample;

FIG. 2 is an enlarged cross-sectional view generally taken along line2-2 of FIG. 1;

FIG. 3 is a top plan view of an exemplary backing member for thepositioning member of FIG. 1;

FIG. 4 is a cross-sectional view of a positioning member in accordancewith an alternative embodiment of the present invention;

FIG. 5 is a perspective view of the assembly of FIG. 1, showing thecarrier engageably mounted on the base mold;

FIG. 6 is a cross-sectional view generally taken along line 6-6 of FIG.5;

FIG. 7 is a perspective view of the base mold of FIG. 1 having thepositioning member disposed therein, showing a front wall of the basemold in an open position;

FIG. 8 is a side elevational view of the assembly of FIG. 5 and aschematic showing a vacuum attachment through the bottom wall of thebase mold;

FIG. 9 is a side elevational view of the assembly of FIG. 5 and aschematic showing a vacuum attachment and fluid port attachment throughthe bottom wall of the base mold;

FIG. 10 is a process flow diagram of the method of the presentinvention;

FIG. 11 is an exploded perspective view of an alternative base moldembodying features of the present invention illustrating an exemplarypositioning member and tissue sample;

FIG. 12 is a top plan view of the base mold of FIG. 11 illustrating anexemplary tissue sample disposed on the positioning member;

FIG. 13 is a cross-sectional view of the base mold of FIG. 12 takengenerally along line 13-13 illustrating an exemplary positioning memberand tissue sample within the base mold;

FIG. 14 is an enlarged, detail view of a portion of the base mold ofFIG. 13;

FIG. 15 is a top plan view of an alternative base mold shown with anexemplary positioning member; and

FIG. 16 is a cross-sectional view of the base mold of FIG. 15 takengenerally along line 16-16.

DETAILED DESCRIPTION OF THE FIRST EMBODIMENT

Referring to FIG. 1, there is illustrated an assembly for in situprocessing of a tissue sample embodying features of the presentinvention. The preferred assembly 10 generally includes a cassette orcarrier member 11 with lid 13, a specimen mounting card or positioningmember 14, and a base mold 12. The assembly 10 allows a tissue sample 15to be initially oriented in a desired position on the specimen mountingcard 14 prior to processing which is maintained throughout subsequentprocessing of the tissue sample 15 and casting in paraffin, or wax. Inthis aspect, the specimen mounting card 14 having the tissue sample 15positioned thereon may be first deposited in the cassette 11 forprocessing and then deposited in the base mold 12 to be cast in a waxblock, with the cassette 11 serving as the base of the block oppositethe tissue sample 15. Tissue processing and casting of the tissue sample15 on the specimen mounting card 14 also may be accomplished within thebase mold 12, and in the alternate method in the snap on holder beneaththe center of the open bottom base mold.

The cassette 11 of the assembly 10 may be any cassette for preparingtissue samples for histological examination known in the art thatprovides an enclosed area for containing a tissue sample duringtransport and processing. Preferably, the cassette should be sized tocooperate with the base mold 12. Exemplary cassettes are as described inU.S. Pat. No. 5,665,398 or 5,928,934, both to McCormick, which arehereby incorporated herein by reference in their entirety; although, itwill be recognized that other types of cassettes could also beeffectively utilized. The cassette 11 generally includes a bottom wall17, a front wall 19, a back wall 21 and two side walls 23.

The specimen mounting card 14 comprises a semi-rigid or rigid, generallyplanar card member. The mounting card 14 is a generally thin,rectangular-shaped card member, with dimensions sized to engageably fitwithin the cassette 11. The specimen mounting card 14 also may besquare-shaped or otherwise configured to generally conform to the shapeand dimensions of the cassette 11. If desired, the top surface 16 of themounting card 14 may be provided with target lines 26 and/or millimeter(or other dimensional) scale markings to provide a measurement scale foridentification and sizing of a sample.

As illustrated in FIG. 2, the mounting card 14 comprises at least onelayer and preferably two or more layers. The first layer 20 includes athin film, such as polypropylene, polytetrafluoroethylene, polyethylene,polyesters and nylon. As an example, the first layer 20 may be Mylar®polyester film by DuPont. The first layer 20 provides a relativelysmooth, non-adhesive surface so that the tissue sample 15 does notadhere to the mounting card 14 to such a degree that it is difficult toremove the wax cast tissue sample 15 in the casting step. Additionally,the top layer 20 is selected so that paraffin generally will not adhereto it in order to facilitate separation of the solidified wax block castfrom the mounting card 14 in the casting step.

Preferably, the film of the first layer 20 is perforated and has aporosity of at least about 15% and no more than about 70% total voidspace, and more preferably at least about 50% and no more than about 60%total void space. A relatively high porosity is desired to allow thedenatured tissue juice (serum) or protein glue to pass through the filmto the layer or layers below, as well as to allow the tissue processingsolvents to pass through the mounting card 14 during processing.

A second layer 22 generally is an absorbent, porous paper. Preferably,the second layer 22 includes an absorbent cellulose paper, such as afilter paper. The second layer 22 preferably has a filter passage ofapproximately 40 to 80% and, more preferably, approximately 60 to 70%,to allow tissue serum, as well as tissue processing solvents, to readilypass through the layer and the specimen mounting card 14.

Generally, the second layer 22 provides the structure to which thetissue sample 15 bonds to the specimen mounting card 14. The tissuesample 15 is mounted on the card 14 as a result of a protein bridge thatforms between the denatured tissue juice, when the tissue is in thefresh state, and the fibers of the paper in the second layer 22.Alternatively, the tissue sample may be mounted to the card 14 as aresult of a protein bridge that forms between a protein glue and thefibers.

To impart increased rigidity to the specimen mounting card 14, thespecimen mounting card 14 may comprise a third layer or backing member24. The third layer preferably comprises a rigid or semi-rigid materialand engageably contacts the second layer 22 at the surface opposite thefirst layer 20. As an example, the third layer 24, may be an acetylmolded backing or a metal backing and may be provided with perforationsor other apertures to allow flow of fluids therethrough. Alternatively,the mounting card 14 may be disposed within a frame of sufficientrigidity that extends around the perimeter of the specimen mounting card14 to hold the layers 20, 22 together and to provide overall rigidity tothe specimen mounting card 14.

As illustrated in FIG. 3, the third layer 24 may comprise a planar metalplate member 110. Preferably, the plate member comprises two opposinglonger side edges 114 a and two shorter opposing side edges 114 bextending between the longer side edges 112 to define a generallyplanar, rectangular surface 116. The surface 116 is provided with aplurality of apertures 118 to allow fluids to pass through the platemember 110. The apertures 118 are positioned across the surface 116 tocreate a predetermined void space in the surface 116. Preferably, theplate member 110 has a void space of at least about 60% and no more thanabout 85% and, more preferably, at least about 70% and no more thanabout 80%. The apertures 118 may be of any suitable dimension andnumber, and may be arranged in any pattern sufficient to achieve thepredetermined void space. The plate member 110 imparts a predetermineddegree of rigidity to the mounting card 14 when it is desired to use aframe, adhesive or other apparatus to maintain the various layers of thespecimen mounting card 14 adjacent one another.

Alternatively, the plate member 110 may be provided with a plurality oftabs, extensions, or bars around its outer perimeter (not shown). Thetabs facilitate the assembly of the mounting card 14 without adhesive ora frame. For example, the layers of the mounting card are positioned atthe top surface of the plate member 110. The tabs are then bent upwardlyaround the outer perimeter of the other layers and the ends are presseddownwardly onto the layers to maintain the layers in engageable contactwith one another and with the plate member 110.

In another embodiment, as illustrated in FIG. 4, the specimen mountingcard 14 comprises a plate member 111 having a paper layer 130 positionedagainst the back surface 132 of the plate member 111, with the paper 130filling the openings 134 of the plate member 111. The paper layer 130may comprise paper pulp slurry which is aspirated, or otherwise applied,onto the back surface 132 of the plate member 111 and into the openings134. The paper layer 130 also may comprise heavy filter paper, or thelike, that is pressed into the back surface 132 and into the openings134. The plate member 111 is constructed of a material selected toprovide, in addition to a sufficiently rigid structure, an essentiallynon-stick surface to which paraffin or other casting material will notadhere. To impart these characteristics, the plate member 111 may beconstructed of a non-reactive metal or alloy, such as stainless steeland the like, or plastic, such as polypropylene, polyethylene and thelike. The plate member 111 may be provided with a skirt 136 that extendsdownwardly around the outer perimeter to provide a cavity into which thepaper layer 130 may be disposed. The plate member 111 also may includetabs or the like (not shown) for securing the paper layer 130 to theback surface 132 of the plate member 111. Alternately the metal platematerial may be coated on one side with a low melt polyethylene and,after a punch perforation, a hot roller application will weld thebacking paper substrate to create a lamination of metal-polyethylenefilm and paper. The adhesion of lamination to paper would be the closedspace of the metal surface.

In this embodiment, the combination of plate member 111 and paper layer130 provide a specimen mounting card 14 onto which the tissue sample 15may be removably mounted for processing. The plate member 111 preferablyhas a void space of at least about 40 and no more than about 80%, morepreferably, at least about 70 and no more than about 85%, and mostpreferably about 65%. The paper layer 130 fills the openings 134 andextends approximately to the upper surface 138 of the plate member 111.

The portions of the paper layer 130 exposed through the openings 134generally provide the structure to which the tissue sample 15, which isdeposited on the upper surface 138 of the plate member 111, bonds, i.e.,the denatured tissue juice from the tissue sample 15 forms a proteinbridge with the paper layer 130 through the openings 134. When it isdesired to remove the tissue sample 15 from the mounting card 14 afterthe tissue sample 15 has been cast in paraffin block, the plate member111 facilitates separation of the cooled paraffin block from thespecimen mounting card 14, because the cooled paraffin generally willnot adhere to the plate member 111.

In another embodiment, which is particularly suitable for smallspecimens (approximately 1 mm thick), the specimen mounting card 14 is aperforated, silicone-treated stiff paper or metal with a thickness ofabout 0.03 mm. The denatured tissue juice, along with the mountant warmprotein or polysaccharide glue, if used, will bond the tissue sample tothe mounting card by gripping the adjacent holes of the perforatedpaper.

Although the mounting card 14 has been described as having two or morelayers of differing materials, it should be understood that the mountingcard 14 may comprise any number and/or combination of layers. Forexample, in another embodiment, the mounting card 14 may comprise onlythe first layer 20 and the second layer 22, with an adhesive, such asacrylic glue, epoxy adhesive, or the like, applied around the perimeterof the mounting card 14 in an amount sufficient to maintain the twolayers 20, 22 in contact with one another, as well as to provide apredetermined degree of rigidity. Alternatively, the mounting card 14may be laminated with the adhesive.

Referring back to FIG. 1, the base mold 12 is generally rectangular inconstruction and is open to the interior surface at the top. The basemold 12 comprises a bottom wall 30, stepped side walls 32, a steppedfront wall 34, and a stepped back wall 36. The base mold 12 isconstructed of a lightweight, rigid material. For example, the base mold12 may be constructed of a relatively inexpensive material, such aspolypropylene, polyethylene, and the like, if the base mold 12 isintended for single use applications. The base mold 12 also may beconstructed of a more durable material, such as a non-reactive metal oralloy, including stainless steel and the like. With more durablematerial, the base mold may be used more than once or connected to anin-line process, which is described below.

The walls 32, 34, 36 each extend upright from the bottom wall 30 adesired height to define a lower recessed chamber 38 at the interior ofthe base mold 12. Preferably, the dimensions of the bottom wall 30 arejust larger than those of the mounting card 14 so that space between thecard 14 and the walls 32, 34, 36 is limited but allows the card 14 to bereadily disposed within the lower chamber 38. The depth of the lowerchamber 38 is at least as high as the thickness of the mounting card 14,to allow the mounting card 14 to be disposed entirely within the chamber38, as best shown in FIG. 6. The walls 32, 34, 36 at the lower chamber38 may be undercut to better hold the mounting card 14 in the desiredplanar position within the lower chamber 38, as well as to retain themounting card 14 when it is desired to separate the wax block withtissue sample 15 embedded therein from the mounting card 14.

From the lower chamber 38, the walls 32, 34, 36 angle outwardly andupwardly, thereby providing increased cross-sectional area in theinterior of the base mold 12. Generally, the side walls 32 projectupwardly at an angle steeper than the angle at which the front and backwalls 34, 36 extend. Above the angled portions, each of the walls 32,34, 36 extend upright to the upper perimeter 40 of the base mold 12 todefine an upper receiving area 48.

Referring to FIGS. 5-7, the upper receiving area 48 preferably has across-sectional area just large enough to receive the cassette 11.Stacking lugs 50 may be provided at the four interior corners where theuppermost upright sections of the walls 32, 34, 36 intersect the angledsections of the walls 32, 34, 36. Alternatively, a bar or otherprojection (not shown) may be provided for support at the side wallsand/or front and back walls for stacking two or more base molds or acassette within a base mold.

A first tab 42 extends generally horizontally from the perimeter 40 atthe front wall 34. A similar second tab 44 extends from the back wall36. Both tabs 42, 44 extend along substantially the width of the frontand back walls 34, 36. The second tab 44 is provided with a fingermember 46 that projects upwardly from the tab 44.

When the cassette 11 is disposed in the base mold 12 at the upperreceiving area 48 (FIG. 5), the finger member 46 engages the back wall21 of the cassette 11 to removably secure the cassette 11. A rib 56(FIG. 7) on the interior surface of the front wall 34 is positioned soas to cooperate with the tab 44 in securing the cassette 11 as describedin U.S. Pat. No. 5,269,671, issued to McCormick, which is incorporatedherein by reference. When the cassette 11 is disposed in the base mold12, the lower edge 58 of the front wall 19 snaps into place below therib 56. The stacking lugs 50 provide additional support for the cassette11 when it is disposed in the receiving area 48.

The front and back walls 34, 36 may be provided with weakened lines 52that extend between the lower chamber 38 and the upper perimeter 40adjacent the edges along which the walls 34, 36 intersect the side walls32. The weakened lines 52 facilitate separation of the front and backwalls 34, 36 so that the walls may be pulled outwardly and downwardlyaway from the base mold 12 (FIG. 7). Deflecting back the walls 34, 36 inthis manner provides increased access to the interior of the base mold12 to facilitate removal of the wax block. An implement may be insertedinto openings 54 in the tabs 42, 44 and used as a lever to break thewalls at the weakened lines 52 and pull back the front and back walls34, 36.

Preferably, the weakened lines 52 do not extend down into the lowerchamber 38. By maintaining the integrity of the walls 32, 34, 36 at thelower chamber 38, the mounting card 14 will be better retained withinthe chamber 38 when it is desired to separate the casting from themounting card 14 and remove the casting from the base mold 12 forfurther processing.

The base mold 12 also preferably comprises outwardly extending feet 60.The feet 60 extend from the walls 32, 34, 36, preferably at the corners.The feet 60 are positioned and configured to provide sufficient supportand stability for the base mold 12.

In another embodiment of the present invention, the base mold 12 isconfigured to allow a vacuum 62 to be drawn from below the bottom wall30, as illustrated in FIG. 8. The bottom wall 30 of the base mold 12 mayinclude perforations or similar openings 61 of sufficient size andspacing to allow the desired vacuum to be drawn. The vacuum pulls themounting card 14 downwardly against the bottom wall 30 to provide aplanar orientation. By drawing a vacuum 62 from below the bottom wall 30as the molten paraffin is poured into the base mold 14, the top layer 20of the mounting card 14 will be pulled taut and flat against the bottomwall 30. With the vacuum being drawn, the wax block casting will beformed on a generally planar surface so as to have few or noimperfections that will result in small pieces of tissue and wax beingsliced separately from the casting.

In another embodiment of the present invention, as illustrated in FIG.9, the base mold 12 may be utilized in an in-line process. A processline 64 is connected to the bottom wall 30 of the base mold 12 forintroduction of solvents into the base mold 12 and subsequent extractionof the solvents. In this embodiment, the solvents for processing thetissue sample 15 are mechanically introduced into the base mold 12 viathe process line 64 instead of being manually introduced from the top.After processing is completed, the solvents are removed from the basemold 12 via the process line 63, which is also connected to the bottomwall 30. In this aspect, the processing of the tissue sample 15 mayoccur via drip extraction. The vacuum 62 may also be connected toprocess line 63 for use either during removal of the process solvents orfor pulling the top layer 20 of the mounting card 14 as described above.Thus, the base mold 12 may also be an essentially self-containedprocessing unit.

Referring to FIGS. 11-16, alternative base mold 112 is illustrated. Thebase mold 112 is similar to base mold 12 with the differenceshighlighted below. In general, the base mold 112 includes stepped sidewalls 132, a stepped front wall 134, a stepped back wall 136, and aremovable bottom wall 130 having a snap-fit connection 140 with at leastone of the walls 132, 134, and 136. Portions of the walls 132, 134, and136 generally form a lower recessed chamber 138 and an upper receivingarea 148. The walls 132, 134, and 136 have bottom edges 144 thatgenerally define a tissue receiving opening 131.

When using the base mold 112, handling of the positioning member 14 isminimized. For instance, the positioning member 14 may be placed on theremoved bottom wall 130, which is then connected to the lower recessedchamber 138 through the snap-fit connection 140. In this manner, thepositioning member 14 with the deposited tissue sample 15 thereon isdirectly inserted into the lower recessed chamber 138 through the tissuereceiving opening 131 rather than the user guiding the positioningmember 14 through the upper receiving area 148 or around walls 132, 134,and 136.

More specifically, as illustrated in FIGS. 11-14, the lower recessedchamber 138 is formed by portions of walls 132, 134, and 136. Forinstance, the lower recessed chamber 138 is generally defined by lowerwall portions 142, which are the lowermost step portions of the walls132, 134, and 136. Each lower wall portion 142 terminates in the bottomedges 144. The size of the lower recessed chamber 138 is determined byintermediate wall portions 146, which are the inner step portions of thewalls 132, 134, and 136. The intermediate wall portions 146 preferablyangle upwardly away from lower wall portions 142. Each intermediate wallportion has a width that generally determines the size of the lowerrecessed chamber 138 and, thus, the tissue receiving opening 131. Forexample, FIGS. 12 and 13 illustrate the base mold 112 with theintermediate wall portions 146 having generally narrower widths todefine a larger, lower recessed chamber 138. Alternatively, asillustrated in FIGS. 15 and 16, the base mold 112 a may have generallylonger intermediate wall portions 146 a to define a generally smallerlower recessed chamber 138 a.

The upper receiving area 148 is generally superimposed above the lowerrecessed area 138. As with the lower area 138, the upper receiving area148 is also defined by portions of the walls 132, 134, and 136. In thisinstance, upper wall portions 150 form the upper receiving area 148. Theupper wall portions 150 are the uppermost steps of the walls 132, 134,and 136 and generally extend upwardly away from the intermediate wallportions 146, preferably in a generally vertical orientation. While notillustrated in FIGS. 11-16, the upper receiving area 148 is sized toreceive the cassette or carrier 11 similar to base mold 12.

Bottom wall 130 preferably defines a cartridge or module 152 sized forreceiving the positioning member 14. Cartridge 152 preferably includesside walls 154 that extend upwardly away from bottom wall 130.Preferably, the dimensions of the cartridge 152 are just larger thanthose of the positioning member 14 so that a space 151 between thepositioning member 14, which is placed in cartridge 152, and thecartridge walls 154 is limited, but allows for the positioning member 14to be readily disposed within the cartridge 152 (FIG. 14). Additionally,the cartridge walls 152 are spaced to correspond to the dimensions ofthe lower recessed chamber 138. As illustrated in FIG. 13, the cartridgewalls 154 are preferably spaced such that the cartridge 152 is slightlywider and longer than lower wall portions 142 of the lower recessedchamber 138. In this manner, the lower wall portions 142 are insertablewithin the cartridge 152. Cartridge 152 may also include flanges 156that extend outwardly from walls 154. Flanges 156 allow a surface forgrasping or handling the cartridge 152 and aid the user in engaging ordisengaging the cartridge 152 into the snap-fit connection 140.

When the cartridge 152 is connected to the base mold 112 through thesnap-fit connection 140, a received positioning member 14 is generallycaptured or secured within the base mold 112. For example, asillustrated in the enlarged cross-sectional view of FIG. 14, a receivedpositioning member 14 is preferably pinched between at least one bottomedge 144 and the cartridge bottom wall 130. In this manner, a peripheraledge portion 153 of the received positioning member 14 is secured withinthe base mold 112 when the cartridge 152 is connected to the lowerrecessed chamber 138.

As illustrated in FIGS. 13 and 14, the snap-fit arrangement 140generally includes a elongated boss or protrusion 158 and acorresponding elongated recess 160. Preferably, the protrusion 158 is inthe lower wall portion 142 and the recess 160 is in the cartridge wall154; however, other arrangements are possible, such as the oppositeconfiguration of the protrusion 158 in the cartridge wall 154 and therecess 160 in the lower wall portion 142. Moreover, one skilled in theart will appreciate that the protrusion 158 and recess 160 may havevarious shapes and sizes so long as the snap-fit connection securescartridge 152 to the lower recessed chamber 138.

To provide the snap-fit arrangement 140, the protrusion 158 seats in therecess 160. In this manner, the seated protrusion 158 secures thecartridge 152 to the lower recessed chamber 138. More specifically, thelower wall portion 142 preferably has an angled leading surface 162,which guides the lower recessed chamber 138 into the cartridge 152.Likewise, cartridge walls 154 also preferably include an angled leadingsurface 164 to further guide the chamber 138 into cartridge 152. To seatthe protrusion 158, cartridge wall 154 preferably has at least one camsurface 166 that pivots either the cartridge wall 154 or the lower wallportion 142 outwardly so that the protrusion 158 may traverse past theleading surface 164 and cam section 166 into the recess 160. Onceseated, the cartridge wall 154 or the lower wall portion 142 pivots backinto its original orientation.

In accordance with the method of the present invention, a tissue sampleis processed for evaluation, as illustrated in FIG. 15. The tissuesample 15 first is collected 70 and is deposited 72 on the mounting card14. The tissue sample 15 is positioned on the mounting card 14preferably immediately after being collected if the entire sample is tobe examined, or once the pathologist or surgeon has selected aparticular aliquot from the tissue sample, so that the orientation ofthe selected plane is preserved throughout processing. Preferably, thetissue sample 15 is deposited with the selected plane disposeddownwardly in contact with the top layer 20 of the mounting card 14. Bythus positioning the tissue sample 15, the selected plane will belocated at the outer face of the wax block after the casting step sothat, when the block is sectioned using the microtome, the selectedplane will be in the leading sections cut from the block.

With a fresh tissue sample, the tissue sample 15 is generally removablymounted on the mounting card 14 as a result of the protein bridge formedwith the second layer 22. If desired, a fixative, such as a proteinglue, may be applied 71 to the outer surface of the top layer 20 priorto mounting of the sample on the card 72 to further adhere the tissuesample 15. Suitable glues, which are compatible with the specimen andwhich do not interfere with contact of the various processing fluids,include proteinaceous adhesives and polysaccharide adhesives. Suitableproteinaceous adhesives include gelatin and casein. Suitablepolysaccharide adhesives include agar, arabinose, carrageenan, andpectin. In general, it is preferred to use proteinaceous adhesives forfresh, non-fixed tissue specimens and to use polysaccharide adhesivesfor previously fixed tissue samples. Additionally, a jellied fixativemay be deposited 74 over the tissue sample 15 which has been positionedon the mounting card 14 to further preserve the integrity of and adherethe tissue sample 15. Suitable jellied fixatives include carbopol orequivalent 0.5% to 1.0% fixative, such as alcohol, formaldehyde,glyoxal, water or 10% solution of formalin.

With the tissue sample 15 mounted on the card 14, the mounting card 14is deposited 76 within the cassette 11. A lid 13 encloses the mountingcard 14 within the cassette. The cassette 11 with mounting card 14 isdeposited in a bag or otherwise packaged in a suitable manner fortransport, as needed.

As shown by process path A, the tissue sample 15 is next subjected totissue processing 78. Solvent processing 78 of the tissue sample 15 maybe completed using equipment and techniques known to one of ordinaryskill in the art. The mounting card 14 remains enclosed in the cassetteduring the processing period and is subjected to a flow of tissuetreating fluids. During processing 78, the tissue sample 15 is contactedwith the desired solvents for dehydrating and clearing the sample.

After processing 78 is completed, the mounting card 14 is removed fromthe cassette 11. The mounting card 14 is next deposited 80 in the basemold 12, 112, or 112 a in the lower chamber 38 in preparation forcasting in wax. The cassette 11 is disposed 82 in the receiving area 48of the base mold 12 above the mounting card 14. When using the base mold12 of the first embodiment of the present invention, the tissue sample15 and mounting card 14 are ready for wax casting. Alternatively, avacuum 84 may be drawn on the base mold 12 prior to and/or during thewax casting step (FIG. 13).

Instead of process path A, process path B can alternatively be usedwhere the tissue sample 15 instead is processed in-line within the basemold 12 (FIG. 14). After the tissue sample 15 has been collected,mounted and transported, the mounting card 14 with tissue sample 15 isremoved from the cassette 11 and deposited 86 in the base mold 12, 112,or 112 a in the lower chamber 38. The cassette 11 is disposed 88 in thereceiving area 48 of the base mold 12 above the mounting card 14. Thebase mold 12 is connected to a solvent process line 64, as describedabove in one of the alternative embodiments, and tissue processing 90 iscommenced. When processing 90 is completed, the process line 64 isdisconnected, and a vacuum line 62 may be connected, if it is desired todraw a vacuum 92.

Next, with all embodiments of the base mold 12, molten embeddingmaterial is introduced 94 through the apertures in the bottom wall 17 ofthe cassette 11 and into the base mold 12. Liquified embedding materialis poured into the mold and around the tissue sample 15 on the mountingcard 14, filling the interior regions of the base mold 14 and thecassette 11, and cooled 96, thereby casting the tissue sample 15 in ablock.

When the tissue sample 15 and cassette 11 are embedded in the block ofsolidified paraffin wax, the block is removed 98 from the base mold 12as previously described. As the block is removed from the base mold 12,the block with tissue sample 15 embedded therein is separated from themounting card 14, which preferably remains in the base mold 12. Thecassette 11 serves as the base of the block opposite the outward,selected face of the tissue sample 15. The sample is then processed perstandard procedures with a microtome.

Numerous modifications to the invention are possible to further improvethe processing of a tissue sample for histological examination. Thus,modifications and variations in practice of the invention will beapparent are expected to those skilled in the art upon consideration ofthe foregoing detailed description of the invention. Although preferredembodiments have been described above and illustrated in theaccompanying drawings, there is no intent to limit the scope of theinvention to these or other particular embodiments.

1. A card member for receiving a tissue specimen to be processed forhistological examination comprising: an absorbent layer for maintainingthe tissue specimen in engageable contact with the card member; agenerally non-adhesive layer located above and opposite the absorbentlayer and in contact with the absorbent layer for allowing separation ofthe tissue specimen located on top of the non-adhesive layer from thecard member; and wherein the card member provides the tissue specimenwith a known, consistent orientation throughout processing.
 2. The cardmember in accordance with claim 1, wherein the tissue specimen is fixedto the absorbent layer incident to formation of a protein bridge betweendenatured tissue juice of the tissue sample and the absorbent portion.3. The card member in accordance with claim 2, wherein a fixativesubstance provides additional adhesion of the tissue specimen to thecard member.
 4. The card member in accordance with claim 1, wherein thecard member has a stiffening layer for increasing rigidity of the cardmember.
 5. The card member in accordance with claim 4, wherein thestiffening layer is a generally planar plate provided with apertures inan upper surface and having a predetermined void space therein forfacilitating passage of fluids therethrough.
 6. The card member inaccordance with claim 5, wherein the void space of the stiffening layeris at least about 60%.
 7. The card member in accordance with claim 5,wherein the stiffening layer includes the non-adhesive layer, whereinthe stiffening layer overlays at least a portion of the absorbent layer,and wherein the apertures of the stiffening layer provide access to theabsorbent layer to facilitate formation of a protein bridge between thedenatured tissue juice and the absorbent layer for removably fixing thetissue specimen to the card member.
 8. The card member in accordancewith claim 7, wherein the stiffening layer includes a material selectedfrom the group consisting of non-reactive metal, non-reactive alloy andplastic.
 9. The card member in accordance with claim 4, wherein thestiffening layer is a generally planar plate having a plurality of tabsat an outer perimeter for maintaining the absorbent portion inengageable contact with the stiffening layer.
 10. The card member inaccordance with claim 1, wherein the non-adhesive layer includes a thinfilm material that is perforated to have a predetermined porosity forproviding passage therethrough to the absorbent layer of an amount ofdenatured tissue juice effective for forming a protein bridge betweenthe juice and the absorbent layer for removably fixing the tissuespecimen.
 11. The card member in accordance with claim 10, wherein theporosity of the thin film is at least about 15%.
 12. The card member inaccordance with claim 10, wherein the thin film material is selectedfrom the group consisting of polypropylene, polytetrafluoroethylene,polyethylene, polyester and nylon.
 13. The card member in accordancewith claim 1, wherein the absorbent layer has a predetermined filterpassage effective for allowing passage of tissue serum and processingfluids therethrough.
 14. The card member in accordance with claim 13,wherein the absorbent layer has a filter passage of between about 60%and about 80%.
 15. The card member in accordance with claim 13, whereinthe absorbent layer has a filter passage of between about 40% and about80%.
 16. The card member in accordance with claim 1, wherein theabsorbent layer includes absorbent porous paper.
 17. The card member inaccordance with claim 16, wherein the absorbent porous paper includesabsorbent cellulose paper.
 18. The card member in accordance with claim16, wherein the absorbent porous paper includes paper pulp slurry. 19.The card member in accordance with claim 17, wherein the absorbentcellulose paper includes filter paper.
 20. A card member for receiving atissue specimen to be processed for histological examination comprising:an absorbent layer for maintaining the tissue specimen in engageablecontact with the card member; a generally non-adhesive layer locatedabove and opposite the absorbent layer and in contact with the absorbentlayer for allowing separation of the tissue specimen located on top ofthe non-adhesive layer from the card member; a backing layer thatengages the absorbent layer located below and opposite the absorbentlayer and in contact with the absorbent layer; and wherein the cardmember provides the tissue specimen with a known, consistent orientationthroughout processing.
 21. The card member in accordance with claim 20,wherein the backing layer is semi-rigid.
 22. The card member inaccordance with claim 21, wherein the semi-rigid backing layer includesmolded acetyl.
 23. The card member in accordance with claim 20, whereinthe backing layer is rigid.
 24. The card member in accordance with claim23, wherein the rigid backing layer includes metal.
 25. The card memberin accordance with claim 20, wherein the backing layer includes a planarmetal plate.
 26. The card member in accordance with claim 25, whereinthe planar metal plate includes a plurality of apertures.
 27. The cardmember in accordance with claim 26, wherein the plurality of aperturesin the planar metal plate creates a void space of at least 60% to about85%.
 28. The card member in accordance with claim 26, wherein theplurality of apertures in the planar metal plate creates a void space ofat least 70% to about 80%.